FIGURE 3:

Exo70p is the only exocyst subunit that can function when anchored to the membrane through a C-terminal transmembrane (TM) domain. (A) The Sso1TM sequence was integrated at the C-terminal end of each exocyst subunit in one of the chromosomal copies in a WT diploid yeast strain. (B) The resulting diploid strains as indicated (NY3251, NY3253-3259) were sporulated and tetrads were dissected onto YPD plates, which were then incubated for 4 d at 25°C. The only viable mutant spores, exo70-2xHA-sso1TM, are marked with circles. (C) WT and exo70-2xHA-sso1TM strains (NY1210 and NY3260) were grown to early log phase in YPD medium at 25°C, serially diluted, spotted onto a YPD plate, and then incubated for 2 d at 25°C. (D) Top panel: WT, sec3Δ, and pADH-sec3-4xHA-sso1TM yeast strains with a complementing SEC3 CEN URA vector (NY3263, 3262, and 3261) were grown to early log phase in SC-UraLeu liquid medium at 25°C, serially diluted, and spotted onto SC-Ura Leu or SC-Leu with 5′-FOA to select against the SEC3 CEN plasmid. The plates were then incubated for 4–5 d at 25°C; bottom panel: WT, pADH-EXO70-2xHA, exo70Δ, and pADH-exo70-2xHA-sso1TM yeast strains carrying a bypass suppressor, 2µ SEC1 URA3 plasmid (NY3267, 3266, 3265, and 3264) were analyzed as described above regarding the top panel. (E) Yeast strains as indicated (NY1210, NY3260, NY3268, NY3269, NY3241) were lysed quickly with NaOH (Materials and Methods). Tagged proteins were detected with anti-HA antibody.